We propose in this application to use breast cell culture systems to examine how specific nutrients and/or combinations of nutrient factors alter gene expression and phenotype, growth characteristics, biomarkers of differentiation and apoptosis. Cells susceptible to transformation as well as transformed cells will be studied. The advantage of using cultured cells is that the levels of polyphenols, vitamins and other media constituents such as hormones and growth factors can be carefully controlled. We will focus our studies on cell culture models of breast preneoplasia and breast cancer. We will simulate the circumstances that breast epithelial cells are under during puberty when estrogen levels rise sharply. We hypothesize that nutrients shown to have chemopreventive properties do so in part by modulating genetic pathways involved in the development of the neoplastic phenotype. Our objective is to evaluate the ability of selected chemopreventive nutrients to inhibit cell proliferation, to attenuate the expression of established biomarkers ofneoplasia, and to modulate genetic pathways. These experiments will exploit the potential of eDNA microarrays and 2-dimensional proteomics. The chemopreventive nutrients that we propose to study are polyphenols and "cancer-protective" vitamins, which likely are contained in all human diets. To test this hypothesis, we propose the following three Specific Aims: 1. To evaluate the influence of polyphenols and vitamins, alone and in combination, on proliferation, DNA damage and anchorage-independent growth of cell models of breast cancer. 2. To determine whether individual and combined polyphenols and vitamins lead to alterations in expression of established biomarkers of breast neoplasia, including DNA methylation. 3. To assess changes in gene and protein expression resulting from growth of preneoplastic and neoplastic breast cells in the presence of polyphenols and vitamins. We hypothesize that combinations of specific polyphenols and vitamins will be more effective than individual components in moderating phenotypic and genotypic changes that occur during the progression of preneoplastic cells to a neoplastic state. The cell culture studies proposed here will provide a comprehensive profile of gene expression changes in human breast cell lines, as a result of exposure of the cells to polyphenols and "cancer-protective" vitamins, two classes of ntrtrients that have been associated with breast cancer prevention.